Thermus aquaticus enzymes

Taq DNA polymerase from Thermus aquaticus) has made it unnecessary to add fresh enzyme for each round of synthesis. Because the amount of target DNA theoretically doubles each round, 25 rounds would increase its concentration about 33 million times. In practice, the increase is actually more like a million times, which is more than ample for gene isolation. Thus, starting with a tiny... 

Tanner, J J., R.M. Hecht, and K.L. Krause. 1996. Determinants of enzyme thermostability observed in the molecular structure of Thermus aquaticus D-glyceralde-hyde-3-phosphate dehydrogenase at 25 Angstroms Resolution. Biochemistry 35 2597-2609. 

Several of the enzymes involved in the processes of repheating, transcription and reverse transcription are available commercially and are used by molecular biologists in the manipulation of nucleic acids. One of the most important of these is Taq polymerase (Taq), which is a thermostable DNA polymerase named after the thermophihe bacterium Thermus aquaticus from which it was originally isolated. This enzyme is especially important, as it is central to the technique known as PCR, which allows sophisticated, targeted in vitro amplification and manipulation of sections of DNA or RNA. DNA... 

The sequence of manipulations in the method is presented in Figure 3.25. An initial problem with the method was that, since the temperature used to separate the strands is about 90 °C, repetitive separation resulted in inactivation of the polymerase, so that fresh enzyme needed to be added for each cycle. The problem was solved by using a DNA polymerase extracted from the organism Thermus aquaticus, which lives in hot springs, so that the enzyme is stable at the high temperature needed to separate the strands. 

PCR makes use of the heat-stable enzyme DNA polymerase from the bacterium Thermus aquaticus and its ability to synthesize complementary strands of DNA when supplied with the necessary deoxyribonu-cleoside triphosphates. We have already looked at the chemistry of DNA replication (see Section 14.2.2), and this process is exactly the same, though it is carried out in the laboratory and has been automated. 

FIGURE 26-4 Structure of the RNA polymerase holoenzyme of the bacterium Thermus aquaticus. (Derived from PDB ID 1 IW7.)The overall structure of this enzyme is very similar to that of the E. coli RNA polymerase no DNA or RNA is shown here. The j3 subunit is in gray, the j3 subunit is white the two a subunits are different shades of red the to subunit is yellow the cr subunit is orange. The image on the left is oriented as in Figure 26-6. When the structure is rotated 180° about the y axis (right) the small to subunit is visible. 

Rather similar ribonucleotide reductases have been isolated from the thermophile, Thermus aquaticus (MW = 80 000) and Anabaena (a blue-green alga) (MW=72 000). The latter enzyme has an absolute requirement for divalent metal cations. The diphosphate reductase from Corynebacterium has a molecular weight of 200 000 and is made up of two subunits. Other enzymes appear to have tetrameric structures.817... 

Aqualysin I is an alkaline serine protease, extracellularly secreted by Thermus aquaticus YT-1,14) an extremely thermophilic, Gram-negative bacterium. The enzyme is thermostable. Optimum temperature for the proteolytic activity of aqualysin I is 80° C in the presence of 1 mM Ca2+ and 70°C in its absence (Fig. 12.1).l5) 85% of the original activity remains after treatment at 80°C for 3 h in the presence of 1 mM Ca2+, but only about 20% remains in its absence.15) These results indicate that calcium ion is essential for the structural stability of the enzyme. 

Terada, Y., Fujii, K., Takaha, T., and Kada, S. 1999. Thermus aquaticus ATCC 33923 amylomaltase gene cloning and expression and enzyme characterization Production of cycloamylose. Appl. Environ. Microbiol., 65, 910-915. 

The heat-stable DNA polymerase (Taq) commonly used in PCR reactions was isolated from a thermophilic bacterium, Thermus aquaticus. Since this enzyme is heat-stable, it can withstand the high temperatures required to denature the DNA template after each successive round of polymerization... 

We describe the various approaches that one might use to generate novel enzymes and the application of one such technique, Random Oligonucleotide Mutagenesis, to the DNA synthesis and repair enzymes DNA polymerase I from E. coli and Thermus aquaticus, as well as human thymidylate synthase, thymidine kinase and 06-alkylgua-nine-DNA alkyltransferase. 

The DNA polymerase enzyme can now extend the primers and complete the replication of the rest of the DNA. The enzyme used in PCR is derived from the thermophilic bacterium Thermus aquaticus, which grows naturally in hot springs at a temperature of 90 °C, so is not denatured by the high temperatures in step 2. Its optimum temperature is about 72 °C, so the mixture is heated to this temperature for a few minutes to allow replication to take place as quickly as possible. 

Thermus aquaticus) and a eukaryote (Saccharoromyces cerevisiae). The two largest subunits for each structure are shown in dark red and dark blue. The similarity of these structures reveals that these enzymes have the same evolutionary origin and have many mechanistic features in common. 

Examples of enzymes suitable for use in molecular biology are the DNA polymerases from Thermococcus //torafa[74] and P. fiiriosus 15 With respect to fidelity of the polymerase reaction both enzymes surpass the DNA polymerase from Thermus aquaticus ( Taq polymerase ) currently used for DNA sequencing and DNA amplification (via PCR reaction). 

The DNA polymerases of T. littoralis and P. furiosus have been marketed for use in DNA amplification by the polymerase chain reaction (PCR) method as the Vent and pfu DNA polymerases, respectively. These enzymes are more accurate in vitro than the Thermus aquaticus (Taq) DNA polymerase, both in classical fidelity tests [132] and in PCR [133,134]. Indeed, they have an associated 3 to 5 exonuclease activity involved in proof-reading, whereas the Taq polymerase is devoid of such activity. 

Xia et have developed an activity-based selection method to evolve DNA polymerases with RNA polymerase activity. Stoffel fragment (SF) of Thermus aquaticus DNA polymerase is displayed on a filamentous phage by fusing it to a coat protein, and the substrate DNA template/primer duplexes are attached to other adjacent coat proteins. Phage particles displaying SF polymerases, which extend the attached primer by incorporating ribonucleoside triphosphates and biotinylated UTP, are immobilized on streptavidin-coated beads. After four rounds of screening a SF library, three mutants were isolated and shown to incorporate ribonucleoside triphosphates virtually as efficiently as the wild-type enzyme incorporates dNTP substrates. 

Polymerases catalyze the synthesis of complementary nucleic acid polymers using a parent strand as a template. In vitro, these enzymes can extend an oligonucleotide primer that is annealed to a template strand. Extension requires that the 3 OH of the extending end is free, and that nucleotide triphosphates (NTPs) are present. Extension stops if you rrm out of template or NTPs, or if no 3 OH groups are. available at the extending end. Thermostable polymerases, such as Thermus aquaticus (Taq) DNA polymerase, are essential reagents for the automation of many nucleic acid amplification procedures. 

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